Carcinogenic and Nephrotoxic Alkaloids Aristolochic Acids upon Activation by NADPH

18 Pages·2010·1.08 MB·English

Gen Physiol Biophys (2001), 20, 375—392 375
Carcinogenic and Nephrotoxic Alkaloids Aristolochic Acids
upon Activation by NADPH : Cytochrome P450 Reductase
Form Adducts Found in DNA of Patients with Chinese
Herbs Nephropathy
M STIBOROVÁ1, M HÁJEK1, E FREI2 AND H H SCHMEISER2
1 Department of Biochemistry, Faculty of Science, Charles University,
Prague, Czech Republic
2 Department of Molecular Toxicology, German Cancer Research Center,
Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
Abstract. Aristolochic acid (AA), a naturally occurring nephrotoxin and carcino
gen, has been found to be implicated in an unique type of renal fibrosis, desig
nated Chinese herbs nephropathy (CHN), and associated with the development of
urothelial cancer in CHN patients Understanding, which enzymes are involved in
A A activation and/or detoxication is important in the assessment of individual sus
ceptibility of humans to this natural carcinogen Using the nuclease PI version of
the 32P-postlabelmg assay we examined the ability of microsomal NADPH CYP
reductase to activate AA to metabolites forming DNA adducts Renal and hep
atic microsomes, containing NADPH CYP reductase, generated AA DNA adduct
patterns reproducing those found in renal tissues in patients suffering from a re
nal fibrosis CHN and urothelial cancel 7-(Deoxyadenosm-N6 yl)anstolactam I, 7-
(deoxyguanosin-N2-yl)anstolactam I and 7-(deoxyadenosm-N6-yl)anstolactam II
were identified as AA-DNA adducts formed by AAI Two AA-DNA adducts, 7
(deoxyguanosm-N2-yl)anstolactam II and 7-(deoxyadenosm-N6-yl)anstolactam II,
were generated from AAII According to the structures of the DNA adducts iden
tified, mtroreduction is the crucial pathway in the metabolic activation of AA The
identity of NADPH CYP reductase as activating enzyme in microsomes has been
proved with different cofactors and an enzyme inhibitor a-Lipoic acid, a selective
inhibitor of NADPH CYP reductase, significantly decreased the amount of the
adducts formed by microsomes Likewise, only a cofactor of the enzyme, NADPH,
supported the DNA adduct formation of AAI and AAII, while NADH was mef
fective These results demonstrate an involvement of NADPH CYP reductase in
the activation pathway of AAI and AAII in the microsomal system Moreover,
using the purified enzyme, the participation of this enzyme m the formation of
AA-DNA adducts was confirmed The results presented here are the first report
Correspondence to Dr Marie Stiborova, Department of Biochemistry, Faculty of
Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic
E-mail stiborovQnatur cum cz
376 Stiborova et al
demonstrating a reductive activation of natural mtroaromatic compounds, AA, by
NADPH CYP reductase
Key words: Aristolochic acid — NADPH cytochrome P450 reductase — Reduc
tive activation — DNA adducts — 32P-postlabelhng — Chinese herbs nephropathy
Abbreviations: AA, aristolochic acid, AAI, 8-methoxy 6-mtro phenanthro-(3,4-
d)-l,3-dioxolo-5-carboxyhc acid, AAII, 6-mtro-phenanthro-(3,4-d)-l,3-dioxolo-5
carboxyhc acid, AAM, a natural mixture of AAI and AAII, AAs, aristolochic acid
I and II, CHAPS, 3-[(3-cholamidopropyl)-dimethylammomo]-l-propane sulfonate,
CHN, Chinese herbs nephropathy, CYP, cytochrome P450, dAp, deoxyadenosme
3'-monophosphate, dGp, deoxyguanosine 3'-monophosphate, dA-AAI, 7-(deoxy-
adenosin-N6-yl)anstolactam I, dA-AAII, 7-(deoxyadenosin-N6-yl)anstolactam II,
dG-AAI, 7-(deoxyguanosin-N2-yl) anstolactam I, dG-AAII, 7-(deoxyguanosm N2-
yl) anstolactam II, HPLC, high performance liquid chromatography, PB, pheno-
barbital, PEI, polyethylemmme, RAL, relative adduct labehng, TLC, thm-layer
chromatography, XO, xanthine oxidase
Introduction
Some of the most potent carcinogens known are natural products (Woo et al 1988)
Among those which have been identified m plants, including safrole, cycasm and
pyrrohzidme alkaloids, aristolochic acids have recently attracted considerable at
tention We reported that aristolochic acid (AA) was implicated m an endemic renal
fibiosis designated Chinese herbs nephropathy (CHN) (Vanherweghem et al 1993),
m young Belgian women who had followed a slimming regimen including Chinese
herbs (Schmeiser et al 1996, Bieler et al 1997, Stiborova et al 1999a, Nortier et
al 2000) An inadvertent replacement of one of these herbs (Stephania tetrandra)
by Anstolochia fangchi has been ascertained by the absence of tetrandrme and
the detection of carcinogenic AA m the herbal powder used for the slimming pills
We detected and identified three AA-specific DNA adducts, 7-(deoxyguanosm-N2-
yl) anstolactam I (dG-AAI), 7-(deoxyadenosm-N6-yl)anstolactam I (dA-AAI) and
7 (deoxyadenosm-N6-yl)anstolactam II (dA-AAII), m renal tissues and thereby
demonstrated that all CHN patients had ingested the plant ingredient AA, the
major alkaloid of anstolochia species AA is a mixture of structurally related mtro-
phenanthrene carboxyhc acids, with 8-methoxy-6-mtro-phenanthro-(3,4-d)-l,3-dio
xolo 5-carboxyhc acid (AAI) and 6-mtro-phenanthro-(3,4-d)-l,3-dioxolo-5-carbo-
xyhc acid (AAII), being the major components (Fig 1) To date over 100 patients
suffering from CHN have been identified (Vanherweghem 1998) A third of the
patients have already received a kidney transplant, another third are receiving
dialysis, and the remainders suffer from slowly progressing renal failure (van Yper
sele de Stnhou 1998) The persistence of the three AA-DNA adducts m human
tissues (kidney and ureter), several months or even years after cessation of the
Activation of Aristolochic Acids by NADPH Cytochrome P450 Reductase 377
Figure 1. Metabolic activation and DNA adduct formation of AAI and AAII 1 R =
OCH, AAI, R = H, AAII, 2 cyclic nitrenium ion of A<U or AAII, 3 R = OCH, ans
3 3
tolactam I, R = H, anstolactam II, 4 R = OCH3, 7-(deoxyadenosm-N6-yl)anstolactam
I (dA-AAI), R = H, 7-(deoxyadenosm-N6-yl)anstolactam II (dA-AAII), 5 R = CH,
3
7-(deoxyguanosm-N2-yl) anstolactam I (dG-AAI), R = H, 7-(deoxyguanosm-N2-yl) ans
tolactam II (dG-AAII)
regimen, is noteworthy (Schmeiser et al. 1996; Bieler et al. 1997; Stiborova et al
1999a, Nortier et al. 2000).
In human tissues the dA-AAI was the most prominent adduct (Schmeiser et
al. 1996; Bieler et al. 1997; Nortier et al 2000) consistent with our previous re
ports on rats given pure AAI or the natural mixture AAM (Stiborova et al 1994)
Both longer persistence and higher initial levels of dA-AAI probably contributed
to the relative abundance of this adduct (Fernando et al 1993; Stiborova et al.
1994; Schmeiser et al. 1996). Moreover, H-ras protooncogenes are activated with
high frequency by an A —> T transversion mutation in codon 61 of DNA from
AAI-induced carcinomas in rats (Schmeiser et al. 1990). This suggests a lelevant
role of the dA-AAI adduct not only in the AA-induced renal fibrotic process, but
also in AA-induced mutagenesis and carcinogenesis. Indeed, an increasing num-
378 Stiborova et al
ber of urothelial carcinomas has been identified in patients with CHN, even after
renal transplantation (Cosyns et al. 1994, 1999; Vanherweghem et al. 1995; Vi-
olon 1997). Therefore, patients with CHN should undergo regular follow-ups for
urothelial cancer.
Recently, Cosyns and coworkers (1999) demonstrated that CHN-dependent
carcinogenesis is associated with the overexpression of p53, which suggests a role
for a mutation in the p53 gene. The relationship between AA-DNA adducts and
p53 mutations in the kidneys and ureters remains to be explored.
Interestingly and luckily, to date only 2-3 % of the patients treated with the
slimming regimen have suffered from nephropathy. Taking into account that AA is
toxic, should it not have affected more of the patients? One possible explanation
for the diffeient responses of patients may be differences in the individual activities
of the enzymes catalyzing the biotransformation (detoxication and/or activation)
of AA. Many enzymes metabolizing carcinogens or their genes are known to exist
in variant forms or show polymorphism that have differing activities. These vari
ations appear to be important determinants of cancer risk (Smith et al. 1995).
Therefore, screening CHN patients as well as healthy women treated with the slim
ming regimen for variations in activities of enzymes involved in AA metabolism or
in genes of them should help to find possible relationships between genotypes or
phenotypes and nephropathy, AA-DNA adduct levels and uiothelial cancer risk.
Thus, the identification of the enzymes principally involved in the activation of
AA in humans and detailed knowledge of their catalytic specificities is of major
importance.
Recently we found that in vitro xanthine oxidase (XO), rat liver microsomal
preparations and even peroxidases were competent in activating both AAI and
AAII to foim the same DNA adducts found m vivo in rodents (Fernando et al.
1993; Stiboiová et al. 1994; Schmeiser et al. 1997) and in humans (Schmeiser et
al. 1996; Bieler et al. 1997; Nortier et al. 2000). The multicomponent micioso-
mal enzyme system consists of NADPH : CYP reductase (EC 1.6.2.4.) and one of
many cytochrome P450 (CYP) isozymes (Rendic and DiCarlo 1997). Both types
of enzymes are integral membiane proteins, and NADPH : CYP reductase is one
of only two known mammalian enzymes containing both FMN and FAD as pros
thetic groups, the other being various isoforms of nitric-oxide svnthase (Wang et al.
1997) Other physiological election acceptors of NADPH : CYP reductase include
microsomal heme oxygenase and cytochrome 6 and although nonphysiological,
5
the reductase is capable of transferring reducing equivalents to cytochrome c and
several exogenous low-molecular-weight substrates (i.e. azo dyes, quinones, nitro-
aromatics) (Wang et al. 1997). NADPH : CYP reductase is present in a membiane
of endoplasmic reticulum of many human tissues including kidney, a target tissue
for CHN. The efficiency of this enzyme to reduce studied natural nitro-aromatics,
aristolochic acids, has not been demonstrated yet. Therefore, the present study
was undertaken to determine the capability of the enzyme to activate these natu
ral products, and to identify whether the enzyme is involved in AA-DNA adduct
formation.
Activation of Aristolochic Acids by NADPH Cytochrome P450 Reductase 379
Materials and Methods
Chemicals and enzymes
Chemicals were obtained from the following sources NADH, NADPH, 3-[(3 chola-
midopropyl) dimethylammomo]-l propane sulfonate (CHAPS), nuclease PI, de
oxyadenosme 3'-monophosphate (dAp), deoxyguanosme 3'-monophosphate (dGp),
dilauroyl phosphatidylcholine, dioleyl phosphatidylcholine and dilauroyl phosphati-
dylserme from Sigma Chemical Co (St Louis, MO, USA), bicmchommc acid
from Pierce, (Rockford, IL, USA) and calf thymus DNA from Roche Diagnos
tics Mannheim (Germany) The natural mixture consisting of 65% AAI and 34%
AAII was a gift from Madaus, Cologne, Germany AAI and AAII were isolated
from the mixture by preparative HPLC (Schmeiser et al 1984) All chemicals were
of analytical purity or better Enzymes and chemicals for the 32P-postlabehng as
say were obtained commercially from sources described previously (Stiborova et al
1994, Schmeiser et al 1997)
Animal experiments
Gavage with AAI and a natural mixture of AA (AAM) dissolved m 0 15 mmol l"1
NaCl (10 mg/kg body weight) was administered to six male Wistar rats (100
150 g) once a day for four consecutive days Six control animals received an equal
volume of 0 15 mmol 1_1 NaCl Rats were placed m cages m temperature- and
humidity-controlled rooms Standardized diet and water were piovided ad libitum
Animals were killed 24 h aftei the last treatment by cervical dislocation (Stiborova
et al 1999b) Liver and kidnej of animals were excised immediately after sacn
fice, quickly frozen in liquid nitrogen, and stored at —80°C until the isolation of
microsomes
Preparation of microsomes, isolation of NADPH CYP reductase and assays
Microsomes from livers and kidneys of control and pre-treated male Wistai rats
weie prepared as described previously (Stiborova et al 1990, 1995) Pre treatment
of six male Wistar rats with phenobarbital (PB) and isolation of microsomes from
lners of these rats were carried out using procedures described by Hodek et al
(1988) Protein concentrations in all microsomal fractions were assessed using
the bicmchommc acid protein assay with serum albumin as a standaid (Wiechel-
man et al 1988) Each microsomal preparation was analyzed foi specific activities
of NADPH CYP reductase The activity of NADPH CYP reductase was mea
sured according to Sottocasa et al (1967) using cytochrome c as the substrate
(i e as N4.DPH cytochrome c reductase) One unit of activity is defined as the
amount of enzyme catalyzing the reduction of 1 //mol of cytochrome c (a, = 21
n
mmol_1l cm^1) per mmute The concentration of NADPH CYP reductase was
estimated as described earlier (Vermilion and Coon 1978) NADPH CYP reduc
tase was purified from rabbit livers as described (Yasukochi et al 1979)
380 Stiborova et al
Incubations
The deaerated and argon-purged incubation mixtures contained in a final volume of
0 75 ml 50 mmol l^1 potassium phosphate buffer (pH 7 4), 1 mmol 1_1 NADPH
1 0 mg of microsomal protein, 0 5 mmol l~x AAI or AAII as sodium salts dissolved
in water and 1 mg of calf thymus DNA (4 mmol 1_1) The íeaction was initiated b}
adding NADPH Unless stated otherwise, incubations were carried out at 37°C for
60 mm The effect of a-hpoic acid (a selective inhibitor of NADPH CYP reduc
tase) (Slepneva et al 1995) was tested using 16 5 0 mmol l_1a hpoic acid Control
incubations were carried out either without activating system (microsomes) or with
activating system, AAI and AAII but without DNA or with activating system and
DNA but without AAs In incubations testing the activity of pure NADPH CV P
reductase, the reaction mixtures were carried out with the enzyme (i) piesent in
liposomes to mimic the conditions of microsomal membrane, and (u) without lipo
some components Briefly, 5 /xmol 1_1 NADPH CYP reductase, 0 5 fig//ú CHAPS,
0 1 fig/(A liposomes [dilauroyl phosphatidylcholine, dioleyl phosphatidylcholine,
dilauroyl phosphatidelsenne (1 1 1)], 3 mmol l"1 reduced glutathione and 50
mmol 1_1 HEPES/KOH, pH 7 4 were mixed in the method (i) In addition, 5
fimol l"1 NADPH CYP reductase and 50 mmol T1 HEPES/KOH, pH 7 4, with
out CHAPS, glutahione and liposomes were mixed and used for incubations in the
method (n) An aliquot containing appropriate amounts of NADPH CYP reduc
tase (0 125 nmol) was added to incubation mixtures instead of microsomes and
used for activation of AAs (see above) After incubation, (37°C, 10 60 mm) the
mixtures were extracted twice with ethyl acetate (2x2 ml) DNA was isolated from
the residual water phase by the phenol/chloroform extraction method as described
earlier (Stiborova et al 1990, 1995, Schmeiser et al 1997) The content of DNA
was determined spectrophotometrically (Schmeiser et al 1997)
32 P-Postlabehng analysis
The nuclease PI enrichment version (Reddy and Randerath 1986) and the
1-butanol extraction-mediated enrichment piocedure (Gupta and Earlj 1988) were
used DNA samples (12 5 fig) were digested with micrococcal nuclease (750 mU)
and spleen phosphodiesterase (12 5 mU) in digestion buffer (20 mmol 1_1 sodium
succinate, 8 mmol l"1 CaCl , pH 6 0) for 3 h at 37°C m a total volume of 12 5 fú
2
2 5 fi\ of the digests were removed and diluted 1 1500 to determine the amount
of normal nucleotides In the nuclease PI version, digests (10 fil) were enriched for
adducts by incubation with 5 fig (5 U) of nuclease PI m 3 fú of a buffer containing
0 8 mol T1 sodium acetate, pH 5 0 and 2 mmol T1 ZnCl for 30 mm at 37°C The
2
reaction was stopped by adding 3 fil of 427 mmol l"1 tns base The extraction with
1-butanol to enrich the adducts was carried out as described earlier (Gupta and
Early 1988) Four microhtres of labeling mix consisting of 400 mmol l^1 biune pH
9 5, 300 mmol l"1 dithiothreitol, 200 mmol T1 MgCl , 10 mmol l"1 spermidine,
2
100 fiCi [7-32P]-ATP (15 pmol), 11 25 Mmol l"1 ATP and 10 U T4 polynucleotide
kinase were added After incubation for 30 mm at room temperature, 20 fil were
Activation of Aristolochic Acids by NADPH Cytochrome P450 Reductase 381
applied to a polyethylenimine (PEI)-coated cellulose TLC plate (Macherey-Nagel,
Diiren, Germany) and chromatographed as described (Schmeiser et al. 1988b) ex­
cept that D3 and D4 were adjusted to pH 4.0 and 9.1 for better resolution. To
determine the amount of normal nucleotides 5 fil of the 1:1500 dilution of digests
were mixed with 2.5 fil of tris buffer (10 mmol-1"1, pH 9.0) and 2.5 fil of labeling
mixture (see above) and incubated for 30 min at room temperature. The labeling
mixture was diluted by mixing 4 fil with 750 fil of 10 mmol-1-1 tris buffer, pH
9.0. 5 fil of this solution were applied to a PEI-cellulose TLC plate and run in
0.28 mol-r1 (NH)S0, 50 mmol-ľ"1 NaHP0, pH 6.5. Adducts and normal
4 2 4 2 4
nucleotides were detected and quantitated by an Instant imager (Packard). Count
rates of adducted fractions were determined from triplicate maps after subtraction
of count rates from adjacent blank areas. Excess of [7~32P]ATP after the postla-
beling reaction was confirmed. Adduct levels were calculated in units of relative
adduct labeling (RAL) which is the ratio of cpm of adducted nucleotides to cpm of
total nucleotides in the assay. Enzymatic synthesis of reference compounds, dAp-
AAI, dGp-AAI, dAp-AAII and dGp-AAII and their 32P-postlabeling were carried
out as described earlier (Stiborova et al. 1994).
Co-chromatography on PEI-cellulose
Adduct spots detected by the 32P-postlabeling assay were excised from the thin-
layer plates, extracted and co-chromatographed with reference 3',5'bisphosphate
adducts as reported previously (Stiborova et al. 1994).
HPLC analysis of 32P-labeled 3',5'-deoxyribonucleoside bisphosphate adducts
HPLC analysis was performed essentially as described previously (Pfau and Phillips
1991; Stiborova et al. 1994; Schmeiser et al. 1997). Individual spots detected by the
32P-postlabeling assay were excised from thin layer plates and extracted (Schmeisei
et al. 1988a). The dried extracts were redissolved in 100 fil of methanol/phosphate
buffer, pH 3.5, 1:1 (v/v). Aliquots (50 fil) were analysed on a phenyl-modified
reversed-phase column (250 x 4.6 mm, 5 fim Zorbas Phenyl; Säulentechnik Di.
Knauer, Berlin, Germany) with a linear gradient of methanol (from 40 to 80 %
in 45 min) in aqueous 0.5 mol-1-1 sodium phosphate and 0.5 mol-1^1 phosphoric
acid, pH 3.5, at a flow rate of 0.9 ml/min. Radioactivity eluting from the column
was measured by monitoring Cerenkov radiation with a Berthold LB 506 C-l flow
through radioactivity monitor (500 fil cell, dwell time 6 s).
Results
We investigated the formation of adducts by AAI and AAII in calf thymus DNA
in the presence of the rat microsomal enzyme systems. Microsomes isolated from
kidneys, a target tissue for AA toxicity, and livers, the tissue rich in a majority
of enzymes metabolizing xenobiotics, were used as model systems. The in vitro
incubations were performed under standardized conditions of AAs (0.5 mmol-1""1),
dNp (4 mmol-1-1) as calf thymus DNA, microsomal protein (1.0 mg) and NADPH
382 Stiborova et al
Figure 2. Autoradiographic profiles of AAI- (A) and AAII-DNA (B) adducts obtained
from DNA after activation by kidney (A, B ), and liver (Aj, B(, ) microsomes The
a a
nuclease Pl-ennchment procedure was used for analysis Origins, in the bottom left-hand
corner were cut off before exposure Screen enhanced autoradiography was at — 80 CC from
2 to 5 h Chromatographic conditions Dl, 1 mol 1_1 sodium phosphate, pH 6 8, D2 was
omitted, D3, 3 5 mol l"1 lithium formate, 8 5 mol ľ"1 urea, pH 4 0, D4, 0 8 mol T1 LiCl,
0 5 mol r1 Tns-HCl, 8 5 mol T1 urea, pH 9 1, D5, 1 7 mol l"1 NaHP0, pH 6 0 Spot
2 4
1, dG-AAI, spot 2, dA-AAI, spot 3, dA-AAII, spot 4, dG-AAII
(1 mmol-1-1) to ensure adduct levels easily detectable by the 32P-postlabeling
method (RAL range 1 adduct in 106-109 nucleotides for enzymatic reactions cat­
alyzed by microsomes). Because enrichment of AA-DNA adducts by n-butanol
extraction yielded a lower recovery (80-90%) as compared to the nuclease PI ver­
sion of the 32P-postlabeling assay, all further experiments were carried out with
enrichment by nuclease PI.
Microsomes of both tissues (kidney, liver) were capable of activating both
AAI and AAII to form DNA adducts. It is evident from Fig. 2 that AAI and
AAII activated by microsomes from these two organs generated the same ma­
jor DNA adduct spots as those obtained m vivo in rats and humans and as re­
ported previously (Schmeiser et al. 1988b, 1996; Stiborova et al. 1994; Bieler et
al. 1997; Nortier et al. 2000). Quantitative analysis (Table 1) revealed that the
Activation of Aristolochic Acids by NADPH Cytochrome P450 Reductase 383
Table 1. Quantitative analysis of adducts formed in DNA by AAI and AAII activated
by liver and kidney microsomal samples
AA-DNA adduct RALa
Liver microsomes Kidney microsomes
AAI-DNA adduct
dG-AAI 5 98 ± 1 046 28 48 ± 4 95c 1 82 ± 0 196 30 33 ± 3 17c
dA-AAI 10 64 ± 1 766 50 67 ± 8 38c 3 17 ± 0 356 52 83 ± 5 83c
dA-AAII 0 83 ± 0 056 3 95 ± 0 23c 0 25 ± 0 036 4 33 ± 0 50c
Total 17 45 ± 1 976 83 10 ± 9 59c 5 25 ± 0 606 87 49 ± 9 53c
AAII-DNA adduct
dG-AAII 0 50 ± 0 156 2 38 ± 0 71c 0 17 ± 0 05fc 2 83 ± 0 83c
dA-AAII 1 58 ± 0 186 7 52 ± 0 86c 0 50 ± 0 106 8 33 ± 1 17c
Total 2 08 ± 0 206 9 90 ± 0 95c 0 67 ± 0 196 11 16 ± 1 83c
Numbers are averages iSEM (n = 6) of triplicate %n vitro incubations, each DNA sam
ple was determined by two post-labeled analyses aRAL, mean iSEM /107 nucleotides,
6RAL, mean iSEM /107 nucleotides per mg protein, CRAL, mean ±SEM /107 nu
cleotides per unit of NADPH CYP reductase
extent of DNA binding by AAI was always higher than by AAII irrespectively
of the type of microsomes used for activation In the organ comparison, micro
somes from livers seemed to be more efficient as an activation system for both
AAs than microsomes from kidneys (Table 1). However, when the levels of AA-
DNA adducts were recalculated and expressed as amounts of adducts per activ
ities of NADPH . CYP íeductase, essentially the same specific levels of adducts
were determined for microsomes of both oigans (Table 1). Control incubations car
ried out in parallel either without microsomes, or without DNA, or without AAs
were free of adduct spots in the region of interest even after prolonged exposure
times. Adduct spots 1, 2 ,3 and 4 formed by AAs (Fig 2) cochromatographed on
PEI-cellulose TLC plates (not shown) and by reversed-phase HPLC (Fig. 3) with
those of synthetic standards (Stiborova et al 1994). Thus, spot 1 was assigned
to 3',5'-bisphospho-7-(deoxyguanosin-N2-yl)-anstolactam I (dG-AAI), spot 2 to
3',5'-bisphospho-7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), spot 3 to 3',5'-
bisphospho-7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) and spot 4 to 3',5'-
bisphospho-7-(deoxyguanosin-N2-yl)-aristolactam II (dG-AAII). These adducts are
known to be generated from AAs by nitro reduction (Pfau et al 1990, 1991, Sti
borova et al. 1994) Therefore, the microsomes tested in this study contain en
zymatic systems capable of catalyzing the reductive activation of AAs leading to
foimation of these DNA adducts. Reductases, NADPH : CYP reductase and NADH
cytochrome 65 reductase, are major candidates for the reductive activation of AAs
by microsomes. To investigate these possibilities, the influences of an inhibitor of
NADPH . CYP reductase and cofactors of both reductases upon AA-DNA adduct
formation catalyzed by microsomes were examined. Rat hepatic microsomes were
used as a model in these experiments.
384 Stiborova et al
dG-AAI dA-AAI o
1 ~
1 1 <NI
\
I
I
\
. .. )
Spot 1 (F.g 2Aa) I1 Spot 2 (Fig 2Aa) 53
22
1 1
1 1
1 l
/V J
10 00 20 00 mm 10 00 20 00 mm
dG-AAII dA-AAII
I-*
\'
ll
I
I \
Spot 4 (Fig 2Ba) Spot 3 (Fig 2Aa, 2Ba) t ^
i
M
ll
I .l/x_A-v
10 00 20 00 10 00 20 00
Figure 3. Separation of 32P-labelled deoxynucleoside 3',5'-bisphosphate adducts derived
from AAI and AAII on a phenyl-modified reversed-phase column Chromatographic con
ditions are described m Materials and Methods Adduct spots were excised and extracted
from PEI-plates, dissolved and injected Standards were obtained from in vitro incuba
tions as described (Stiborova et al 1994) For clarity, HPLC profiles are shown m arbitrary
units
As shown in Table 2 the formation of AA-DNA adducts had an absolute re
quirement for NADPH, a known cofactor of NADPH: CYP reductase. Adduct
levels were negligible when NADPH was omitted from the incubation mixture